Automatic processing system for use in solid phase biospecific binding and DNA sequencing techniques

ABSTRACT

A system for processing biomolecules comprising, a plurality of manifolds, each manifold having a holder portion and a number of peg members extending therefrom, a plurality of plate or strip means, each supporting a number of wells adapted to receive the manifold peg members, a processing device which comprises several processing units in the form of at least one heating plate unit adapted to receive the well plate or strip means for heating the wells thereof, at least one wash unit having a plurality of wash wells, each wash well being adapted to receive one or more manifold peg members and having at least one inlet and at least one outlet for wash liquid, and a holder device for holding a plurality of manifolds during processing in and transfer between different units of the processing device.

The present invention relates to a system for processing biomolecules,particularly in DNA sequencing.

Laboratory processes involving nucleic acid analyses are nowadays commonand are often performed as a routine. Such processes include inter aliahybridization and enzymatic reactions. Often a solid phase is used forimmobilization of one component of an interaction. In many casesmagnetic beads have been used as the solid phase due to the simplifiedseparation of such a solid phase. Simultaneous handling of a largenumber of samples has, however, still been a problem.

Our international patent application with publication number WO 94/11529discloses a solid phase technique for nucleic acid analysis which highlyfacilitates the simultaneous processing of a large number of samples.This technique is based on the use of a "multi-peg" device, preferablyin the form of a comb element, where each peg or tooth forms a separatesolid phase member. The combs are designed to be introduced with theirteeth into specially designed well plates or strips, the wells of whichcontain the necessary reagents.

The present invention relates to a complete processing system based onsuch comb and well members and comprising components specially designedfor cooperation therewith, which system further facilitates theperformance of nucleic acid analyses as well as other types ofbiomolecular interactions or analyses and makes possible thesimultaneous handling and processing of a large number of samples. Sucha system has the features given in the claims and is described in moredetail below with regard to a non-limiting embodiment designed fornucleic acid analysis.

Reference is made to the accompanying drawings, wherein:

FIG. 1 is a front view of a carrier comb;

FIG. 2 is a top view of the comb in FIG. 1;

FIG. 3 is an assembly of eight combs shown in FIG. 1;

FIG. 4 is a top view of one type of well plate;

FIG. 5 is a top view of another type of well plate;

FIG. 6 is a side view of the well plates shown in FIGS. 4 and 5,respectively;

FIG. 7 is a top view, with parts broken away, of a processing unit;

FIG. 8 is a side view of the processing unit in FIG. 7;

FIG. 9 is another side view of the processing unit in FIG. 7;

FIG. 10 is a sectional view through A--A in FIG. 7;

FIG. 11 is a top view of a comb holder;

FIG. 12 is a sectional view through A--A in FIG. 11 and a lid member;

FIG. 13 is a top view of a well plate holder; and

FIG. 14 is a side view of the well plate holder in FIG. 13.

FIG. 15 is a top view of a positioning module.

The processing system shown in the figures basically comprises a carriercomb 10 (FIG. 1), two types of well plates 20 and 30 (FIGS. 4 and 5), aprocessing unit 40 (FIGS. 7 to 10) and a comb holder 60 (FIGS. 11 and12). Also included in the illustrated system is a well plate holder 80(FIGS. 13 and 14) and a positioning module (FIG. 15).

The flat comb 10 has eight teeth 11 adapted to be received in wells 21and wells 31 of well plate 30, respectively. Comb 10 is further providedwith coupling means, here in the form of two cylindrical protrusions 12with open bottoms and a tip with reduced diameter which will fit intothe open bottom of an aligned comb 10. Thereby several combs may besnapped together as illustrated in FIG. 3, which shows an assembly ofeight combs 10. A top flange 13 is provided on comb 10 for labellingpurposes, such as by optically readable bar codes. Each side edge of thecomb 10 has a step portion 14 to permit the comb to be held in combholder 60 as will be described below.

Well plate 20 has, in the illustrated case, eight rows of wells 21, eachrow of which has two wells. Each well 21, which may be dimensioned tohold, e.g., a liquid volume of about 100 μl, is designed to receive fourcomb teeth 11 and is therefore hereinafter sometimes referred to as"four teeth well". Similarly, well plate 30 has eigth well rows, eachrow having eight wells 31 designed to receive a single well comb tooth11, and well type 31 is therefore hereinafter sometimes referred to as"one tooth well". Each well 31 may, e.g., be dimensioned to hold aliquid volume of about 20 μl.

The integral processing unit shown in FIGS. 7 to 9 comprises a chassis41 in which, in the illustrated, five processing modules or "stations"are mounted, viz. a "capture" station 42, a "denaturation" station 43, a"wash" station 44, an "annealing" station 45, and a "sequencingreaction" station 46.

The capture station 42 is a plate for thermal control, i.e. heating orcooling, with four groups of eight vertical heating or cooling flanges47. Each heating flange 47 has a central recess for receiving one row ofwells 21 of a well plate 20, such that four well plates may be placed onthe heating plate simultaneously with the wells 21 in thermal contactwith the heating flanges.

The denaturation station 43 has a similar structure to the capturestation 42 and also comprises a plate with four sets of eight flanges 48for receiving a respective well plate 21. The denaturation station 43may or may not be heatable.

Wash station 44 comprises, as is best shown in FIG. 10, a plate 49 withfour groups of eight elongated recesses 50. Each recess 50 is adapted toreceive the eight teeth 11 of a comb 10. Means are further provided forpumping wash liquid separately through each recess, e.g., from a centralinlet to outlets at the ends of each recess, in the illustrated case twopumps 51, 52 for inlet and outlet, respectively. The illustrated washstation 44 may thus process 32 combs 10 simultaneously.

Annealing station 45 has a similar structure to the denaturation station43 and comprises a plate with four sets of eight flanges 53, each setadapted to receive a well plate 21. In the same way as for the capturestation 42, means are provided for controlled heating and cooling offlanges 53.

The sequencing reaction station 46, finally, is also a heating platewith four sets of eight recesses 54. Each recess 54 is designed toreceive one row of well plates 31. The illustrated design of heatingplate, with recesses 54 provided in a solid plate rather than in heatingflanges as in stations 42, 43 and 45 described above, may be used inthis station since no rapid temperature changes are required here, aswill be described below.

Reference numeral 55 indicates a free "set aside" area.

Reference numerals 56 at stations 42-46 indicate light signal means, thefunction of which will be described below.

Processing stations 43, 45 and 46 all have a central lock member 57 forcooperation with the comb holder 60 as will be described below. Further,all the processing stations 42-46 have four protrusions 58 for aligningthe comb holder 60 therewith, as will also be described below.

The processing unit 40 is adapted to be connected to a computer for thecontrol of the different processing stations, such as heating and washliquid flow, and operation of the unit is performed via a small key padand display 59.

Comb holder 60 shown in FIGS. 11 and 12 is designed for the transfer ofcombs 10 between the different processing stations. Holder 60 comprisesa horizontal frame 61 attached to a handle 62. A comb rack 63 is mountedinside frame 61 to be vertically movable in relation to the frame. Combrack 63 is here formed by a rectangular frame 64 with a central crossbar 65 normal to handle 62. Recesses 66 in the cross bar 65 and theopposite frame parts are designed to receive and hold combs 10 via thestepped vertical side edges 14 thereof (FIG. 1). Four comb holdingsections, each with eight opposed recesses 66, are defined by the crossbar 65 and the handle 62, each section thus being capable of holdingeight combs.

Comb rack 63 is supported by a vertical bar 67 extending through a bore68 in handle 62. The upper end of bar 67 is in turn rotatably connectedto a knob member 69 received in a recess 70 in the handle. A verticalplate-like extension 71 of the knob member 69, when aligned with therecess, fits into the upper part of bore 68 and may be received therein,as is shown in FIG. 12, the comb rack 63 thereby being held a first,lowered position. In this position, the teeth 11 of each supported comb10 are inserted into a corresponding well 21 or 31 of a well plate 20 or30, respectively, placed on any one of the processing stations when thecomb holder 61 is properly placed on that station. By lifting knobmember 69, and thereby comb rack 63, and then turning the knob to placethe extension 71 out of alignment with its recess, the comb rack will beheld in a second, raised position.

The comb holder 60 is further provided with two opposed spring-biasedhook means 72, each having an operation lever 73, for cooperation withpin members 81 on the well plate holder 80, as will be described below.

An exemplary use of the above described processing system for DNAsequencing of PCR amplified DNA fragments to be sequenced will now bedescribed.

Four well plates 20 with PCR amplified biotinylated DNA fragments to besequenced, one strand of which is biotinylated, are supported by thewell plate holder 80. Each well 21 may contain a different DNA fragment.

Comb holder 60 is loaded with combs 10, to the teeth 11 of whichstreptavidin has been immobilized, the comb rack 63 being held in theraised position. The comb tops may be covered by two lids 74 (FIG. 12)placed over the combs on either side of the handle 62. The comb holder60 is then placed correctly on the well plate holder 80 so that the hookmeans 72 engage with the pin members 81, and the whole assembly is thenmoved to capture station 42 of the processing unit. The combs 10 havepreviously been provided with bar codes, and these are now read into thesystem computer to relate each comb to the amplification product from aparticular sample. The indicator light 56 flashes to indicate that thisstation is to be used, and the display 59 may, e.g., indicate "Go toposition one".

Next, the comb holder 60 is aligned with the capture station 42 andplaced thereon so that the protrusions 58 fit into recesses 75 in theholder frame 61. The well rows of the well plates 20 will be received inthe recesses of the heating flanges 47. The comb rack 63 is then loweredto enable the comb teeth 11 of each comb 10 to be introduced into thecorresponding wells 21. When the comb rack is in the lowered position,an activator member 76 supported by the comb rack contacts the chassis41 to effect a switch function starting the capture process. To thisend, activator member 76 may, for instance, contain a magnet whichactivates an aligned Hall element mounted in the chassis. The captureprocess, in which biotinylated DNA fragments are bound to thestreptavidin coated comb teeth, may, for example, advantageously consistof two heating cycles 20°-80° C. completed during 30 minutes. Suchcycled heating will enhance the capture process. During the captureprocess, the light indicator 56 emits a steady light and the display mayshow the station number and remaining process time. When thepredetermined process time has passed, which may be indicated by adiscrete sound signal, the capture function will stop. Simultaneously,the light signal stops. Instead the light signal indicator 56 at thedenaturation station 43 will start to flash and and the display may,e.g., show "Go to position 2".

The comb holder 60 is then released from the well plate holder 80 byraising the comb rack 63 to the upper position and pressing the levers73 of hook means 72 towards each other to disengage them from the pinmembers 81, whereupon the comb holder is moved to the denaturationstation 43.

Well plates 20, which have the necessary denaturation agent in the wells21 thereof for release of the non-biotinylated DNA-strand from the DNAfragments just captured on the comb teeth, have previously been placedon the denaturation station 43 with the well rows received in therespective heating flange recesses. Preferably, the well plates havebeen prepared in advance with predispensed anhydrated reagents, e.g.glassified, so that only water need to be added before use.

The comb holder 60 is then aligned with and placed on the denaturationstation 43, and the comb rack is lowered to insert the comb teeth 11into the respective wells 21. The lower end of the bar 67 will thenengage with the lock member 57 to prevent removal of the comb holder 60if the comb rack has not been raised to its upper position where thecomb teeth are above the wells.

As before, the light indicator 56 will have a steady light during theprocess, which may last for, e.g., 5 min. When this time has passed, thelight will go out, a sound signal buzz, and the display may show, e.g.,"Go to position 3".

In the same was as described above, the comb holder 60 is then moved tothe wash station, where the comb teeth are washed for, e.g., 3 min.

Comb holder 60 is then transferred to the annealing station 45 wherewell plates 20 with sequencing primer solutions in the wells 21 havebeen placed on the heating flanges 53. Preferably, the wells 21 havebeen predispensed with anhydrated primer mixes so that only water isadded before use. Annealing of primers to the single stranded DNAfragments immobilized on the comb teeth may, for example, be performedby heating at 60° C. for 10 min and then controlled cooling for 10 minto room temperature.

The comb holder 60 is finally moved to the sequencing reaction station46, where well plates 30 with "one tooth wells" have been placed on theheating plate with the wells 31 received in the recesses 54. The wells31 contain the necessary sequencing mixes, preferably predispensed inanhydrated form to only require the addition of water at the time ofuse.

When the sequencing reactions are completed, the combs 11 are brought toan automated sequencer, e.g., an A.L.F.™ DNA Sequencer (PharmaciaBiotech AB, Uppsala, Sweden) where the primer extension products on thecomb teeth are released into the sample wells of the sequencer.

As a further embodiment, a positioning module, A in FIG. 15, can beincluded in the system for processing biomolecules according to theinvention. The positioning module, A, is situated before the processingmodules or "stations" shown in FIG. 7 and described above. In FIG. 15one such "station" is indicated with B. The positioning module consistsof a metal plate with holes designed for receiving the wells of amicro-titre plate. The size of the metal plate is that of standardmicro-titre format, both concerning the number of wells as well as thedistance between the wells. In the bottom of each hole there is alight-emitting diode. When a micro-titre plate has been placed on thepositioning module it is possible to see through the bottom of each wellif the light-emitting diode is switched on. This principle is used toshow from which well the sample shall be brought. The processing moduleor "station", B, shown in FIG. 15 comprises four well plates of the typeshown in FIG. 4. Each well plate has 2×8 wells 21 and at the side ofeach well there is a light-emitting diode 82.

When the positioning module is used a micro-titre plate containingsamples of e.g. PCR amplified DNA fragments is placed on the positioningmodule A. Four empty well plates are placed on the processing module B.The computer programme controlling the positioning is activated and someof the light-emitting diodes are swithed on. By means of thelight-emitting diodes on the positioning module and on the processingmodule respectively, and the displayed-information text on the displaypanel, information is obtained from which position on the positioningmodule to which position of the processing module the sample shall bemoved. A common operation is to move samples from one horizontal row inthe micro-titre plate to a horizontal row in the well plate by means ofa 8-canal pipette.

As is readily seen from the above, the described processing systemprevents mix-up of samples, prevents contamination between samples andis easy to operate.

The invention is, of course, not restricted to the embodimentsspecifically described above and shown in the drawings, but many changesand modifications may be made without departing from the inventiveconcept as defined in the following claims.

I claim:
 1. A system for processing biomolecules, comprising:a pluralityof manifolds (10), each manifold having a holder portion and a number ofpeg members (11) extending thereform, a plurality of plate or stripmeans (20, 30), each supporting a number of wells (21, 31) adapted toreceive the manifold peg members (11), a processing device (40) whichcomprises a plurality of processing modules (42-46) in the form of(i) atleast one module for thermal control (42) adapted to receive the wellplate or strip means (20, 30) for heating or cooling the wells (21, 31)thereof, (ii) at least one wash module (44) having a plurality of washwells (50), each wash well (50) being adapted to receive one or moremanifold peg members (11) and having at least one inlet and at least oneoutlet for wash liquid, and a holder device (60) for holding a pluralityof manifolds (10) during processing in and transfer between differentmodules of the processing device (40), and computer means forcontrolling the function of the processing device (40), and indicatormeans (56) controlled by said computer means to indicate when and onwhich processing module (42-46) each processing step in a sequence ofprocessing steps is to be performed.
 2. The system according to claim 1,characterized in that said manifolds are comb elements (10) with teeth(11).
 3. The system according to claim 1 or 2, characterized in that theholder device (60), when placed on a processing module (42-46), isarranged to hold the manifolds (10) in two alternative verticalpositions above a well plate or strip (20, 30) received on the module, alower position in which the manifold peg members (11) are inserted intothe wells (21, 31), and an upper position in which the peg members (11)are above the wells (21, 31).
 4. The system according to claim 1,characterized in that the holder device (60) comprises activator means(74) which, when the holder device (60) is placed on a processing module(42-46) and the manifolds (10) are brought to the lower position,activate start of the process at the processing module (42-46).
 5. Thesystem according to claim 1, characterized in that each manifold (10) orgroup of manifolds are labelled by optically readable labels foridentification of different manifolds (10).
 6. The system according toclaim 1, characterized in that the system is adapted for sequencing ofnucleic acid fragments and comprises:a first, heatable plate module (42)for performing capture of nucleic acid fragments on manifold peg members(11), a second plate module (42), which optionally may be heated, forperforming denaturation of captured double stranded nucleic acidfragments into immobilized single stranded nucleic acid fragments, awash module (44) for washing manifold peg members (11) which have beensubjected to the denaturation step, a third, heatable plate module (45)for performing annealing of sequencing primers to single strandednucleic acid fragments immobilized to manifold peg members (11), and afourth, heatable plate module (46) for performing sequencing reactionson the primer annealed nucleic acid fragments immobilized to manifoldpeg members (11).
 7. The system according to claim 1, characterized inthat the system comprises well plates or strips (20, 30) where the wells(21, 31) contain predispensed reagents in anhydrated form.
 8. The systemaccording to claim 1, characterized in that the manifolds (10) areprovided with coupling means (12) for enabling coupling of two or moremanifolds (10) to form an assembly thereof.
 9. The system according toclaim 5, wherein the optically readable labels are bar codes.